CN113092414B - SARS-CoV-2 antibody detection method - Google Patents

Abstract

The invention discloses an unmarked detection method of a novel coronavirus antibody. The method of the invention achieves the detection purpose by modifying an anti-SARS-CoV-2 antibody on the end face of the optical fiber, combining the SARS-CoV-2 antibody in a sample with the SARS-CoV-2 antibody on the end face of the optical fiber, increasing the refractive index at the end face of the optical fiber due to the specific combination of the two antibodies, and detecting the change of the light intensity in the optical fiber. The method has the advantages of extremely small required sample amount, simple operation and high accuracy, and can be used for specifically detecting two new crown antibodies.

Description

A kind of detection method of SARS-CoV-2 antibody

technical field

The invention belongs to the field of biotechnology, and in particular relates to a detection method for SARS-CoV-2 antibodies.

Background technique

The novel coronavirus (SARS-CoV-2) is highly contagious and pathogenic, and spread rapidly after the outbreak, causing global public health problems. Timely and accurate detection of viral nucleic acid and antibodies is of great significance to the control of the epidemic. At present, the commonly used methods for the detection of the new coronavirus (SARS-CoV-2) mainly include: viral nucleic acid real-time polymerase chain reaction method, enzyme-linked immunoassay method, chemiluminescence immunoassay method, flow immunoassay method, etc.

Viral nucleic acid real-time polymerase chain reaction method is a common method for clinical diagnosis of patients with new coronavirus, known as the "gold standard". However, due to the sampling process and sample preservation problems, the test results often produce unavoidable " false negatives" and "false positives". The serological detection of anti-SARS-CoV-2 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies induced by the new coronavirus infection has high sensitivity, specificity and stability, and can be used as an effective nucleic acid detection method. complementary approach. Commonly used detection kits for novel coronavirus (SARS-CoV-2) antibodies, such as enzyme-linked immunoassay (ELISA), chemiluminescent enzyme immunoassay (CLIA) and flow immunoassay (LFIA) kits, can be used for specific detection SARS-CoV-2 antibodies. Among them, enzyme-linked immunosorbent assay (ELISA) detection has lower requirements for equipment, does not require large-scale equipment, and can realize the quantitative detection of new crown antibodies, but the operation steps of this method are relatively cumbersome and the detection cycle is long; chemiluminescence enzyme immunoassay Analysis (CLIA) can also realize the quantitative detection of the new crown antibody, but this method requires expensive and complicated instruments and professional operators; although flow immunoassay (LFIA) can quickly obtain the detection results, this method can only be qualitative or semi-antibody. Quantitative analysis of antibody concentration cannot accurately detect antibody concentration. Therefore, it is urgent to develop a rapid, simple and sensitive serological detection technology for clinical diagnosis of new coronavirus pneumonia.

Contents of the invention

The purpose of the present invention is to provide a label-free detection method for novel coronavirus antibodies, which is more convenient, accurate and traceable to realize the serological immunity of SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG). Quantitative detection method to solve the shortcomings and deficiencies of the existing chemiluminescent enzyme immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) to detect new crown antibodies, such as time-consuming and complicated operation. The amount of sample required by the invention is very small, the operation is simple, and the accuracy is high, and the specific detection of two new coronavirus antibodies can be carried out.

In a first aspect, the present invention provides a functionalized fiber optic probe.

The functionalized optical fiber probe provided by the present invention is prepared according to the method comprising the following steps:

a) Using a multimode optical fiber as a detection probe, removing the coating layer on the surface of the optical fiber, and grinding and polishing both ends of the optical fiber;

b) Place the end face of the optical fiber in a piranha solution of V(H ₂ SO ₄ ):V(H ₂ O ₂ )=3:1 for reaction, and wash and dry the optical fiber with ultrapure water after the reaction;

c) Soak the end face of one end of the optical fiber in step b) in a toluene solution of MTS (methyl p-toluenesulfonate), wash and dry after the reaction;

d) Soak the end face of the optical fiber treated in step c) in GMBS (4-maleimidobutyric acid-N-succinimidyl ester) ethanol solution, and clean the optical fiber for later use after the reaction.

e) Soak the end face of the optical fiber processed in step d) in the solution of anti-SARS-CoV-2 antibody (ie, secondary antibody, which is a specific antibody against SARS-CoV-2 antibody), and react overnight in a refrigerator at 4°C. Clean the optical fiber with ultrapure water after the end;

f) Seal the unreacted site on the end face of the optical fiber with BSA solution for 2 hours, and make a SARS-CoV-2 IgG functionalized detection optical fiber or a SARS-CoV-2 IgM functionalized detection optical fiber.

In step a) of the above method, the diameter of the multimode optical fiber may be 400-600 microns, and the numerical aperture of the optical fiber may be 0.18-0.22. The length of the optical fiber may be 3-5 cm.

In step b) of the above method, the reaction conditions are: heating at 70° C. for 20-40 minutes.

In step c) of the above method, the mass concentration of MTS in the MTS toluene solution is 1%-5% (specifically, 2%); the soaking time may be 1-3 hours.

In step d) of the above method, the concentration of GMBS in the ethanol solution of GMBS (4-maleimidobutyric acid-N-succinimidyl ester) can be 10-50mM (specifically, 20mM); the soaking The time can be 0.5-2 hours.

In step e) of the above method, the concentration of the anti-SARS-CoV-2 antibody in the anti-SARS-CoV-2 antibody solution may be 100 μg/mL-1000 μg/mL. The anti-SARS-CoV-2 antibody can be an anti-SARS-CoV-2 IgG antibody or an anti-SARS-CoV-2 IgM antibody.

In step f) of the above method, the mass concentration of the BSA solution is 1%-5%. The blocking time is 2-3 hours.

The functionalized optical fiber probe prepared by the above method also belongs to the protection content of the present invention.

In the second aspect, the present invention also protects the application of the above-mentioned functionalized fiber optic probe.

The application of the functionalized fiber optic probe provided by the present invention is its application in the preparation of a kit for detecting SARS-CoV-2 antibodies.

The SARS-CoV-2 antibody can be a SARS-CoV-2 IgG antibody and/or a SARS-CoV-2 IgM antibody.

In the third aspect, the present invention also protects the application of the functionalized fiber optic probe and the SARS-CoV-2 antibody standard.

The application of the functionalized optical fiber probe and the SARS-CoV-2 antibody standard provided by the present invention is their application in the preparation of a kit for detecting the SARS-CoV-2 antibody.

The SARS-CoV-2 antibody can be a SARS-CoV-2 IgG antibody and/or a SARS-CoV-2 IgM antibody.

In the fourth aspect, the present invention also protects a kit for detecting SARS-CoV-2 antibodies.

The kit for detecting SARS-CoV-2 antibodies provided by the present invention includes the functionalized fiber optic probe provided by the present invention.

Further, the kit for detecting SARS-CoV-2 antibodies also includes SARS-CoV-2 antibody standards.

Further, the kit for detecting the SARS-CoV-2 antibody also includes a carrier on which the following method for detecting the SARS-CoV-2 antibody is recorded.

In the fifth aspect, the present invention also protects a method for detecting the antibody content of SARS-CoV-2.

The method for detecting SARS-CoV-2 antibody content provided by the present invention comprises the following steps:

c1) Passing PBS buffer solution to the surface of the functionalized optical fiber probe prepared above for 1-2 minutes to stabilize the light signal and obtain a reflected light intensity signal value of 1;

c2) Pass SARS-CoV-2 antibody standards of different concentrations into the optical fiber end face of the functionalized optical fiber probe, react for 5-7 minutes, and obtain a reflected light intensity signal value of 2;

c3) Then pass the regenerating solution to the surface of the optical fiber for 3-5 minutes, so that the SARS-CoV-2 IgG antibody dissociates from the anti-SARS-CoV-2 IgG antibody on the surface of the functionalized fiber optic probe, and finally pass to the surface of the optical fiber The detection result of PBS buffer in 1-5 minutes is signal value 1-signal value 2;

c4) Repeat the steps of c1)-c3) above, measure the reflected light intensity signal value 2 corresponding to the SARS-CoV-2 antibody standard product with a series concentration, and obtain the detection results corresponding to the SARS-CoV-2 antibody standard product with a series concentration, to The concentration of the SARS-CoV-2 antibody standard substance is the abscissa, and the standard curve is drawn by Logistic fitting with the detection result signal value corresponding to the concentration of the SARS-CoV-2 antibody standard substance on the ordinate to obtain the standard curve equation;

c5) The sample to be tested is detected according to the method in the above c1)-c3), and the signal value of the test result corresponding to the sample to be tested is obtained, and it is substituted into the standard curve equation obtained in c4), and the α in the sample to be tested is calculated. Concentrations of SARS-CoV-2 antibodies.

The above method can be used to detect SARS-CoV-2 IgG antibody or SARS-CoV-2 IgM antibody.

When detecting SARS-CoV-2 IgG antibody, the optical fiber end face of the functionalized fiber optic probe has anti-SARS-CoV-2 IgG antibody;

When detecting SARS-CoV-2 IgM antibody, the optical fiber end face of the functionalized fiber optic probe has anti-SARS-CoV-2 IgM antibody.

In the above method, the regeneration solution is an aqueous solution of SDS with a mass concentration of 0.5%.

The sample to be tested in the present invention may specifically be serum to be tested. Before the test, the serum to be tested needs to be diluted 20-50 times with PBS buffer, and placed at 4°C for use.

The present invention has the following advantages due to the adoption of the above technical scheme: 1. The novel coronavirus (SARS-CoV-2) antibody detection method of the present invention, by modifying the anti-new coronavirus (SARS-CoV-2) antibody at the end face of the optical fiber, The new coronavirus (SARS-CoV-2) antibody in the sample is combined with the new coronavirus (SARS-CoV-2) antibody on the end face of the optical fiber, and the refractive index at the end face of the optical fiber increases due to the specific combination of the two antibodies. Detect the change of light intensity in the optical fiber to achieve the purpose of detection. 2. The new coronavirus (SARS-CoV-2) antibody detection method of the present invention has a fast detection speed and can specifically and quantitatively detect the content of the new coronavirus antibody.

Description of drawings

Figure 1 is a typical curve diagram for detection of novel coronavirus SARS-CoV-2 IgG;

Figure 2 The standard curve for the detection of novel coronavirus SARS-CoV-2 IgG.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with specific implementation cases, so that those skilled in the art can better understand the invention and implement it, but the examples given are not intended to limit the present invention.

The case cited this time is the spiked detection of IgG antibodies against novel coronavirus in human serum samples.

The SARS-CoV-2 IgG antibody standard used in the following examples was purchased from Beijing Boaolong Immunology Technology Co., Ltd. BF02020.

The anti-SARS-CoV-2 IgG antibody used in the following examples was purchased from Beijing Boaolong Immunology Technology Co., Ltd. BF03082-S.

The multimode fiber used in the following examples was purchased from RMDX-23 of Beijing Shouliang Technology Co., Ltd.

Example

The detection method of novel coronavirus (SARS-CoV-2) IgG antibody provided by the invention comprises the following steps:

(1) Fabrication of functionalized fiber optic probes

a1) A multimode optical fiber with a diameter of 600 microns is used as the detection probe, the numerical aperture of the optical fiber is 0.22, the coating layer on the surface of the optical fiber is removed, the length of the removed coating layer is 5 cm, and the total length of the optical fiber probe is 5 cm. Ends ground and polished.

b1) Place the end face of the optical fiber in a piranha solution of V(H ₂ SO ₄ ):V(H ₂ O ₂ )=3:1, heat at 70°C for 30 minutes, and wash the optical fiber with ultrapure water after the reaction Dry clean.

c1) Soak the end face of one end of the optical fiber in step b1) in 2% MTS (methyl p-toluenesulfonate) toluene solution for 2 hours, wash and dry after the reaction.

d1) Soak the end face of the optical fiber treated in step c) in 20mM GMBS (4-maleimidobutyric acid-N-succinimidyl ester) ethanol solution for 1 hour, and clean the optical fiber after the reaction spare.

e1) Soak the end face of the optical fiber treated in step d) in an anti-SARS-CoV-2 IgG antibody solution with a concentration of 100 μg/mL and react overnight in a refrigerator at 4° C. After the reaction, clean the optical fiber with ultrapure water.

f1) Seal the unreacted sites on the end face of the optical fiber with a BSA solution with a mass concentration of 2% for 2 hours to make a SARS-CoV-2 IgG functionalized detection optical fiber.

(2) Establishment of standard curve

Get the SARS-CoV-2 IgG antibody standard substance that gradient concentration is 10, 20, 50, 100, 500, 1000ng/mL to detect, and the detection process is specifically: to step (1) prepared " anti-SARS-CoV-2 IgG The surface of the antibody functionalized detection fiber is passed into PBS buffer for 60 seconds to stabilize the light signal and obtain a reflected light intensity signal value of 1, and then pass the new coronavirus IgG antibody standard (that is, SARS-CoV-2 IgG antibody standard product), reacted for 300 seconds, and obtained a reflected light intensity signal value of 2. Pass into regeneration solution (mass concentration 0.5% SDS aqueous solution) to optical fiber surface then 300 seconds, make SARS-CoV-2 IgG antibody and the secondary antibody on optical fiber surface (secondary antibody is the specific antibody against SARS-CoV-2 IgG antibody, That is, the aforementioned anti-SARS-CoV-2 IgG antibody) is dissociated, and finally the surface of the optical fiber is passed into PBS buffer solution for 75 seconds to clean the surface of the optical fiber. The test result is signal value 1-signal value 2, and the real-time typical curve of the test is shown in Figure 1. Different concentrations of SARS-CoV-2 IgG antibody standards were sequentially injected to obtain a SARS-CoV-2 IgG antibody detection standard curve as shown in Figure 2.

(3) Sample testing

Add 25, 50, and 100 ng/mL IgG antibodies to the sera of 6 different healthy people for detection. The specific detection steps are as follows:

a2) Dilute the serum to be tested 20 times with PBS buffer, and store it at 4°C for later use.

b2) Pass PBS buffer solution to the surface of the optical fiber for 60 seconds to stabilize the optical signal and obtain a signal value of 1, then pass diluted serum to be tested to the end face of the optical fiber, react for 300 seconds, obtain a signal value of 2, and then pass it to the surface of the optical fiber The regeneration solution (0.5% SDS aqueous solution) was washed for 300 seconds, and finally PBS buffer solution was passed through the surface of the optical fiber for 75 seconds to restore the optical signal to the original value.

c2) The test result is "signal value 1 minus signal value 2". Compare the test result with the established SARS-CoV-2 IgG detection standard curve to obtain the concentration of SARS-CoV-2 IgG in the serum to be tested. The results are shown in the following table :

The above results show that the recovery rate of SARS-CoV-2 IgG antibody spiked is 95.2%-105.2%, the relative standard deviation is less than 10%, and the detection result is reliable.

The detection limit is defined as the lowest detected concentration, and the linear range of detection is obtained from the established standard curve. The detection limit of the standard curve shown in the attached figure is 5 ng/mL, and the linear range is 10.025-109.393 ng/mL.

The detection principle of the present invention is as follows:

Light is transmitted in the optical fiber. Because the end face of the fiber is smooth and flat enough, and because the refractive index inside the fiber and outside the end face of the fiber are different, the light undergoes Fresnel reflection at the end face of the fiber, and the light reflected back to the fiber can be detected and reflected back to the fiber The light intensity is related to the refractive index at the end face of the fiber, and the greater the refractive index at the end face of the fiber, the smaller the light intensity reflected back to the fiber. The anti-new coronavirus antibody is modified on the end face of the optical fiber. The new crown antibody contained in the sample to be tested can specifically bind to the anti-new crown antibody on the end face of the optical fiber. The refractive index at the end face of the optical fiber increases due to the specific combination of the two antibodies. As the light decreases, the refractive index changes caused by different concentrations of antibodies are different, and the reflected light intensity at the end face of the optical fiber is also different. The purpose of quantitatively detecting the concentration of the new coronavirus antibody in the serum sample can be achieved by detecting the change of light intensity.

The above are only preferred implementation cases of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the specification and content of the present invention, or directly or indirectly used in other related technical fields, are the same as The theory is included in the patent protection scope of the present invention.

Claims (10)

1. A preparation method for a functionalized fiber optic probe, comprising the steps of: a) Use multimode optical fiber as the detection probe, remove the coating on the surface of the optical fiber, and grind and polish both ends of the optical fiber; b) The end face of the optical fiber is placed in the piranha solution for reaction, and after the reaction is completed, the optical fiber is washed and dried with ultrapure water; c) Soak the end face of one end of the optical fiber in step b) in a toluene solution of methyl p-toluenesulfonate, wash and dry after the reaction; d) Soak the end face of the optical fiber treated in step c) in an ethanol solution of 4-maleimidobutyric acid-N-succinimidyl ester, and clean the optical fiber for later use after the reaction; e) Soak the end face of the optical fiber treated in step d) in the anti-SARS-CoV-2 antibody solution, react overnight in a refrigerator at 4°C, and clean the optical fiber with ultrapure water after the reaction; f) Sealing unreacted sites on the end face of the optical fiber with BSA solution to make the functionalized optical fiber probe. 2. The preparation method according to claim 1, characterized in that: in the step a), the diameter of the multimode optical fiber is 400-600 microns, the numerical aperture of the optical fiber is 0.18-0.22; the length of the optical fiber is 3-5 cm; In the step b), the piranha solution is a mixed solution of V(H ₂ SO ₄ ):V(H ₂ O ₂ )=3:1; In the step b), the reaction conditions are: heating at 70°C for 20-40 minutes; In the step c), the mass concentration of methyl p-toluenesulfonate in the toluene solution of methyl p-toluenesulfonate is 1%~5%; the soaking time is 1-3 hours; In the step d), the amount of 4-maleimidobutyric acid-N-succinimide ester in the ethanol solution of 4-maleimidyl butyric acid-N-succinimide ester The concentration is 10-50mM; the soaking time is 0.5-2 hours; In the step e), the concentration of the anti-SARS-CoV-2 antibody in the anti-SARS-CoV-2 antibody solution is 100 μg/mL-1000 μg/mL; the anti-SARS-CoV-2 antibody is an anti-SARS-CoV -2 IgG antibody or anti-SARS-CoV-2 IgM antibody; In the step f), the mass concentration of the BSA solution is 1%-5%; the blocking time is 2-3 hours. 3. The functionalized fiber optic probe prepared by the method according to claim 1 or 2. 4. the application of functionalized optical fiber probe according to claim 3 in the test kit for preparing detection SARS-CoV-2 antibody. 5. the application of functionalized optical fiber probe and SARS-CoV-2 antibody standard item in the preparation detection kit of SARS-CoV-2 antibody described in claim 3. 6. The application according to claim 4 or 5, characterized in that: the SARS-CoV-2 antibody is a SARS-CoV-2 IgG antibody and/or a SARS-CoV-2 IgM antibody. 7. A test kit for detecting SARS-CoV-2 antibodies, comprising the functionalized fiber optic probe of claim 3. 8. The kit according to claim 7, characterized in that: the kit for detecting SARS-CoV-2 antibodies also includes a SARS-CoV-2 antibody standard; Further, the kit for detecting the SARS-CoV-2 antibody also includes a carrier on which the method for detecting the content of the SARS-CoV-2 antibody according to claim 9 or 10 is recorded. 9. A method for detecting SARS-CoV-2 antibody content for non-diagnostic purposes, comprising the steps of: c1) passing PBS buffer solution to the surface of the functionalized optical fiber probe according to claim 3 for 1-2 minutes, so that the light signal is stable, and the reflected light intensity signal value is 1; c2) Pass SARS-CoV-2 antibody standards of different concentrations into the optical fiber end face of the functionalized optical fiber probe, react for 5-7 minutes, and obtain a reflected light intensity signal value of 2; c3) Then pass the regeneration solution to the surface of the optical fiber for 3-5 minutes, so that the SARS-CoV-2 IgG antibody dissociates from the anti-SARS-CoV-2 IgG antibody on the surface of the functionalized fiber optic probe, and finally pass PBS to the surface of the fiber optic Buffer for 1-5 minutes, the detection result is signal value 1-signal value 2; c4) Repeat the above steps c1)-c3), measure the reflected light intensity signal value 2 corresponding to the SARS-CoV-2 antibody standard product with a series concentration, and obtain the detection results corresponding to the SARS-CoV-2 antibody standard product with a series concentration, and use The concentration of the SARS-CoV-2 antibody standard substance is the abscissa, and the standard curve is drawn by Logistic fitting with the detection result signal value corresponding to the concentration of the SARS-CoV-2 antibody standard substance on the ordinate to obtain the standard curve equation; c5) The serum sample to be tested is tested according to the method in c1)-c3) above, and the signal value of the test result corresponding to the serum sample to be tested is obtained, and then substituted into the standard curve equation obtained in c4), and the sample to be tested is calculated and obtained Concentrations of SARS-CoV-2 antibodies in . 10. The method according to claim 9, characterized in that: the method is used to detect SARS-CoV-2 IgG antibody or SARS-CoV-2 IgM antibody; When detecting SARS-CoV-2 IgG antibody, the optical fiber end face of the functionalized fiber optic probe has anti-SARS-CoV-2 IgG antibody; When detecting SARS-CoV-2 IgM antibody, the optical fiber end face of the functionalized fiber optic probe has anti-SARS-CoV-2 IgM antibody; The regeneration solution is a 0.5% SDS aqueous solution with a mass concentration; Before testing, the serum to be tested needs to be diluted 20-50 times with PBS buffer solution, and stored at 4°C for use.

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